The long term objective of these studies is to understand the molecular structure of the tight junction so that its functional properties can be manipulated for therapeutic purposes. Tight junctions (TJ) form a continuous intercellular seal between both epithelial and endothelial cells, creating a barrier to the paracellular movement of water, solutes such as therapeutic drugs, and immune cells. The TJs barrier properties are quite variable among cell types and physiologically regulated, although the molecular basis of this remains unknown. Pathologic processes like inflammation and specific bacterial toxins alter barrier properties with significant consequences for epithelial function. Although several TJs proteins have been identified, their functions remain unknown and several more proteins remain to be identified. This lack of knowledge has greatly impeded formulation of ideas about how cellular signaling processes regulate the paracellular barrier. The barrier appears to depend on the organization of perijunctional actin. The specific aims of this application are to define the structural organization and function of the tight junction protein ZO-1. Recent cDNA sequencing of human ZO-1 reveals it to be a member of the Membrane-Associated Guanylate Kinase protein family (MAGUK). These are multidomain proteins found at specialized sites of cell-cell contact and speculated to serve both structural and signaling roles. One MAGUK, the product of the Drosophila discs-large gene is a known tumor suppressor. We will define the sequences within ZO-1 which bind to the known TJ proteins, ZO-2 and occludin. Methods will include the yeast two-hybrid system and co-immunoprecipitation of truncated fragments of ZO-1 expressed in cultured cells through transfection techniques. Binding interactions will be correlated with sequences within ZO-1 responsible for TJ targeting in cultured cells and for inducing changes in perijunctional actin. We hypothesize that ZO-1 influences the organization of actin and this is a mechanism that regulates paracellular permeability. Cell lines used include the MDCK canine kidney and HepG2 hepatocyte and Caco-2 enterocyte human cell lines. New TJ proteins which bind to ZO-1 will be sought using yeast two-hybrid screening of a human liver cDNA library.